A targeting construct was designed to insert a loxP/frt-flanked neomycin (neo) resistance cassette downstream of exon 4 of the X-linked myotubular myopathy gene 1 (Mtm1) gene. A c.205C-T point mutation was introduced in exon 4, corresponding to human amino acid 205, resulting in the missense mutation, p.R69C, commonly found in humans with X-linked myotubular myopathy (MTM). This targeting construct was electroporated into C57BL/6 x 129/SvEv-derived embryonic stem (ES) cells and correctly targeted ES cells were injected into C57BL/6J blastocysts. The resulting chimeric mice were bred to C57BL/6J mice. The donating investigator reports mice were backcrossed to C57BL/6J mice for at least ten generations (see SNP results below) prior to sending males to The Jackson Laboratory Repository in 2012. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the first generation rederived living colony at The Jackson Laboratory Repository. While the 26 markers throughout the somatic genome suggested a C57BL/6 genetic background, all 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.
